8OSC

Structure of Homo sapiens 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 (DNPH1) bound to deoxyuridine 5'- monophosphate

8OSC の概要

• エントリーDOI: 10.2210/pdb8osc/pdb
• 関連するPDBエントリー: 8OS9
• 分子名称: 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, 2'-DEOXYURIDINE 5'-MONOPHOSPHATE (3 entities in total)
• 機能のキーワード: deoxyribonucleoside 5'-monophosphate n-glycosidase activity, hydrolase
• 由来する生物種: Homo sapiens (human)
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 32696.62

構造登録者

Devi, S.,da Silva, R.G. (登録日: 2023-04-18, 公開日: 2023-08-30, 最終更新日: 2023-09-13)

主引用文献

Devi, S.,Carberry, A.E.,Zickuhr, G.M.,Dickson, A.L.,Harrison, D.J.,da Silva, R.G.
Human 2'-Deoxynucleoside 5'-Phosphate N -Hydrolase 1: Mechanism of 2'-Deoxyuridine 5'-Monophosphate Hydrolysis.
Biochemistry, 62:2658-2668, 2023

PubMed Abstract: The enzyme 2'-deoxynucleoside 5'-phosphate -hydrolase 1 (DNPH1) catalyzes the -ribosidic bond cleavage of 5-hydroxymethyl-2'-deoxyuridine 5'-monophosphate to generate 2-deoxyribose 5-phosphate and 5-hydroxymethyluracil. DNPH1 accepts other 2'-deoxynucleoside 5'-monophosphates as slow-reacting substrates. DNPH1 inhibition is a promising strategy to overcome resistance to and potentiate anticancer poly(ADP-ribose) polymerase inhibitors. We solved the crystal structure of unliganded human DNPH1 and took advantage of the slow reactivity of 2'-deoxyuridine 5'-monophosphate (dUMP) as a substrate to obtain a crystal structure of the DNPH1:dUMP Michaelis complex. In both structures, the carboxylate group of the catalytic Glu residue, proposed to act as a nucleophile in covalent catalysis, forms an apparent low-barrier hydrogen bond with the hydroxyl group of a conserved Tyr residue. The crystal structures are supported by functional data, with liquid chromatography-mass spectrometry analysis showing that DNPH1 incubation with dUMP leads to slow yet complete hydrolysis of the substrate. A direct UV-vis absorbance-based assay allowed characterization of DNPH1 kinetics at low dUMP concentrations. A bell-shaped pH-rate profile indicated that acid-base catalysis is operational and that for maximum /, two groups with an average p of 6.4 must be deprotonated, while two groups with an average p of 8.2 must be protonated. A modestly inverse solvent viscosity effect rules out diffusional processes involved in dUMP binding to and possibly uracil release from the enzyme as rate limiting to /. Solvent deuterium isotope effects on / and were inverse and unity, respectively. A reaction mechanism for dUMP hydrolysis is proposed.

PubMed: 37582341
DOI: 10.1021/acs.biochem.3c00369

実験手法

X-RAY DIFFRACTION (1.42 Å)